Most immune responses occur only if a T lymphocyte recognizes foreign antigen in association with a self class II molecule of the major histocompatibility complex (MHC). Antigen must be processed for presentation by class II molecules and this processing generally involves endocytosis of antigen into an acidic compartment where newly synthesized class II molecules have been transported. The foreign peptide/MHC class II complexes recognized by CD4 T cells are thus formed intracellularly. The aim of this project is to define the potential sources of antigen for presentation by class II molecules, and to determine how class II molecules are transported to antigen-processing compartments. A genetic approach was used to demonstrate that several processing pathways exist for antigen presentation by class II molecules. The classical pathway for presentation of exogenous antigen requires newly synthesized class II molecules and a function encoded by an unknown gene within the class II region of the MHC. However, presentation of the influenza virus hemagglutinin antigen was independent of protein synthesis and of the function encoded by the unknown gene, implying that two processing pathways exist. In addition, it was demonstrated that two endogenous processing pathways lead to the presentation of cytosolic antigen to class II-restricted T cells, one is dependent on the transporter for antigen presentation (TAP), the other is not. In addition, the TAP-independent pathway requires another function that maps within the class II region of the MHC. This previously unsuspected presentation of cytosolic proteins by class II molecules has important implications for T cell repertoire selection, T cell tolerance, autoimmunity and vaccine design. Targeting of newly synthesized class II molecules to endosomes is mediated by the invariant chain (Ii), but the intracellular transport route is not known. This project demonstrated that a large population of class II-Ii complexes reach endosomes by rapid internalization from the cell surface. The highly efficient endocytosis was mediated by the cytoplasmic tail of Ii. Following internalization, Ii dissociated from class II-Ii complexes, suggesting that this pathway represent an important mechanism for loading class II molecules with peptides from several endocytic compartments.